1-lower alkyl or alkylene substituted-6,7-methylenedioxy-4(1h)-oxocinnoline-3-carboxylic acids and methods for making and using same

ABSTRACT

1-Lower alkyl or alkenyl substituted-6,7-methylenedioxy-4(1H)oxocinnoline-3-carboxylic acids, active against Gram-negative bacteria and Mycoplasma organisms and methods for the preparation and use thereof.

United States Patent White June 13, 1972 Inventor: William A. White,Fountaintown, lnd.

Assignee: Eli Lilly and Company, Indianapolis, Ind.

Filed: Dec. 29, 1969 Appl. No.: 888,880

Related U. S. Application Data Continuation-impart of Ser No. 796,546,Feb. 4,

1969,21bandoned.

US. Cl. ..260/250 A, 424/250, 260/340.5 meg! ..C07d5l/08 Field of Search..260/25O [56] References Cited OTHER PUBLlCATlONS Chemical Abstracts,Vol. 60, page 9,344- 9,345 (1964). Chemical Abstracts, Vol. 69, page3,365 1968 Primary Examiner-Nicholas S. Rizzo Att0rney-Everet F. Smithand Walter E. Buting [57] ABSTRACT l-Lower alkyl or alkenylsubstituted-6,7-methylenedioxy- 4(1l-l)-oxocinnoline-3-carboxylic acids,active against Gramnegative bacteria and Mycoplasma organisms andmethods for the preparation and use thereof.

19 Claims, N0 Drawings CROSS REFERENCE This application is acontinuation-in-part of my copending application, Ser. No. 796,546,filed February 4, 1969 now abandoned.

BACKGROUND OF THE INVENTION l-leretofore, a number of compounds havebeen used to combat microbial infections arising from Gram-negativebacteria and Mycoplasma organisms. Among such compounds are nalidixicacid and the substituted quinolines described in US. Pat. No. 3,287,458.None of the prior art compounds is efiective against all strains ofGram-negative bacteria or Mycoplasma or a mixed combination of the two.Therefore, the search for more effective antimicrobials is a continuingone.

SUMMARY New therapeutic compositions have now been discovered that areefiective against a broad spectrum of microbial organisms. These newantimicrobial compositions comprise certain l-lower alkyl or alkenylsubstituted-6,7-methylenedioxy-4(l H)-oxocinnoline-3-carboxylic acids,prepared from 2-nitro-4 ',5-methylenedioxyacetophenone by a series ofreactions involving hydrogenation to the 2'-amino compound,diazotization, and ring closure to the corresponding cinnoline-4-ol,bromination to the 3-bromocinnoline derivative, reaction with cuprouscyanide to prepare the 3-carbonitrile, introduction of a methyl, ethyl,n-propyl, isopropyl, n-butyl, or allyl group into the l-position withsimultaneous tautomerization of the hydroxyl at the 4-position to the(lI-I)-oxo, and hydrolysis of the carbonitrile group to a carboxylicacid group. A number of novel compounds are obtained as intermediates inthis synthe- SIS.

These new antimicrobial compounds can be employed in the form of thefree acids or as nontoxic cationic salts of such acids for theprevention and treatment of microbial infections.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS The novel antimicrobialcompounds of this invention are 1- lower alkyl or alkenyl substituted-6,7-methylenedio 4gl H )-oxocinnoline-3-carboxylic acids having thestructure:

wherein R is a member of the group consisting of methyl, ethyl,n-propyl, isopropyl, n-butyl, vinyl and allyl.

The term lower alkyl or alkenyl" as used herein means methyl, ethyl,n-propyl, isopropyl, n-butyl, vinyl and allyl.

The novel compounds of the present invention can be prepared by amulti-step synthesis starting with the known compound, 2 '-nitro-4,5'-methylenedioxy-acetophenone. The foregoing starting material isreduced, utilizing platinum dioxide as a catalyst, in a Parrhydrogenation shaker at about 50 psi at a temperature of about 30-40 C.to form the new compound, 2'-amino-4',5'-methylenedioxyacetophenone.Those skilled in the art will recognize that other catalysts, such aspalladium on carbon, or Raney Nickel, can also be employed at pressuresand temperatures other than those specified; or that the reduction canalso be effected by using a suitable chemical reduction process.

The second step of the reaction sequence comprises diazotizing and ringclosing the amino compound to a cinnoline derivative by diazotizing theaminoacetophenone with an equimolar amount of a nitrous acid-generatingnitrite in the presence of a fiveto ten-fold molar excess of a solutionof hydrochloric acid at a temperature of 0 C. or below, then ringclosing over a period of 3-24 hours at a temperature of 0-80 C., thepreferred reaction conditions being 3-4 hours at C. The reaction yieldsthe known 6,7-methylenedioxycinnoline- 4-ol [Schofield and Simpson, J.Chem. Soc., 519 I945 of the formula:

The cinnoline derivative can be brominated with bromine in the presenceof an approximately equimolar amount of potassium acetate in a fivetoten-fold molar excess of acetic acid at the refluxing temperature of thereaction mixture. The resulting new compound,3-bromo-6,7-methylenedioxycinnoline-4- ol, is converted to6,7-methylenedioxycinnoline-4-ol-3-carbonitrile by treating the bromoderivan've with from about an equimolar amount to about a twofold molarexcess of cuprous cyanide. The reaction is generally conducted in afiveto 20- fold molar excess of dimethylformamide at atmosphericpressure for fi'om 4-24 hours at a temperature of from about 50 C. tothe refluxing temperature of the reaction mixture, preferably atrefluxing temperatures of 4-6 hours.

Alkylation of 6,7-methylenedioxycinnoline-4-ol-3-carbonitrile with amethyl, ethyl, n-propyl, isopropyl, n-butyl or allyl bromide, chloride,iodide or sulfate can be eflected in accordance with known procedures ina polar organic solvent such as a C, to C alkanol, a C to C, alkanediol,diethyleneglycol, triethyleneglycol, dimethylformamide,dimethylacetamide, N-methylpyrrolidone, and the like, in the presence ofa basic reagent to produce the corresponding 1- methyl, ethyl, n-propyl,isopropyl, n-butyl, or alkyl-6,7- methylenedioxy 4( lI-I)-oxocinnoline-3-carbonitrile compounds of this invention.

The final step in the synthesis comprises hydrolysis of the carbonitrileto the corresponding carboxylic acid. The hydrolysis can be convenientlycarried out with either an acid or a base. The preferred processcomprises hydrolysis with concentrated hydrochloric acid in a reactionmedium of acetic acid wherein the hydrochloric acid and the acetic acidare present in a molar ratio of from 1.4 to 4:1 and the concentration ofthe acetic acid relative to the nitrile is in a molar ratio offrom 5:1to 20:1.

l-Vinyl-6,7-methylenedioxy-4( l I-I)-oxocinnoline-3carboxylic acid isprepared by alkylating 6,7-methylenedioxy-cinnoline4-ol-3-carbonitrilewith l-bromo-2-hydroxyethane to forml-(B-hydroxyethyl)-6,7-methylenedioxy-4(lI-I)-oxocinnoline-B-carbonitrile and hydrolyzing the alkylatedcarbonitrile to the corresponding carboxylic acid as described above.The l-(B-hydroxyethyl)-6,7-methylenedioxy-4( 1H)-oxocinnoline-Bcarboxylic acid is then esterified using ptoluene-sulfonylchloride in pyridine to form the tosylate. Elimination of the tosylateusing a strong base, such as sodium ethoxide in anhydrous ethanol,yields the desired l-vinyl-6,7- methylenedioxy-4(lH)-oxocinnoline-3-carboxylic acid. Other suitable methods for effectingthe elimination of the elements of water from the B-hydroxyethylsubstituent can also be employed. A variety of such methods are known inthe art.

Also included within the scope of this invention are the salts of theabove-described l-lower alkyl or alkenyl substituted-6,7-methylenedioxy-4( lH)-oxocinnoline-3-carboxylic acids with thealkali metals such as lithium, sodium and potassium; the alkaline earthmetals such as magnesium, calcium, strontium, and barium; and ammonium;and salts of amines having a pKa greater than 7.5 such as, for example,triethylamine, pyrrolidine, and the like. The salts can be formed byreacting the free acid with a suitable base, basic salt or amine such assodium bicarbonate, calcium carbonate, ammonium hydroxide,triethylamine, and the like. Among the preferred salts are the sodium,potassium, calcium, and ammonium salts.

The 1 -substituted-6,7-methylenedioxy-4( lHD-oxocinnoline-3 carboxylicacids and their salts are essentially white, high-melting crystallinesolids. The free acids are soluble in most polar organic solvents,whereas the monovalent cationic salts are soluble in aqueous solvents.

This invention is further illustrated by the following examples:

EXAMPLE 1 Preparation of 2-Amino-4,5'-Methylenedioxyacetophenone About90 gm. (0.43 mole) of 2'-nitro-4',5'-methylenedioxyacetophenone weresuspended in about 300 ml. of absolute ethanol and 200 mg. of platinumdioxide were added. The mixture was placed in a Parr hydrogenationshaker and reduced with hydrogen at 50 psi at a temperature of 3040 C.for 3-4 hours until no further hydrogen uptake was observed. The solventwas evaporated from the reaction mixture under vacuum and the residuewas taken up in 500 ml. of hot chloroform. The mixture was filtered andthe filtrate was evaporated to dryness. The residue was recrystallizedfrom an 80:20 mixture of ethanol and water to yield about 70 gm. ofyellowish-brown crystals of 2-amino-4,5-methylenedioxyacetophenonehaving a melting point of about l67l68 C. The yield was 90.7 percent.

Analysis: C H NO Mol. Wt.: 179;

Calc.: C, 60.33; H, 5.06; N, 7.82;

Found: C, 60.60; H, 5.33; N, 7.75.

EXAMPLE 2 Preparation of 6,7-Methylenedioxycinnoline-4-ol About 70 gm.(0.41 mole) of 2'-amino-4',5'- methylenedioxyacetophenone were added to800 ml. of concentrated hydrochloric acid at room temperature. Theresultant mixture was cooled to C. and a solution of 35 gm. (0.5 mole)of sodium nitrite in 100 ml. of water was added dropwise while thetemperature was maintained at 0 C. with stirring. Stirring was continuedfor 30 minutes after the sodium nitrite addition was completed. Theresultant reaction mixture was filtered and the filtrate was warmed to80 C. for 4 hours, allowed to cool to room temperature overnight toeffect crystallization of the product, and filtered. The crystals soobtained were washed with water and then recrystallized from 600 ml. ofan 80:20 mixture of dimethyl sulfoxide and water. About 65 gm. of6,7-methylenedioxycinnoline-l-ol as light tan crystals were obtained.The product melted at about 338 C. with decomposition. The yield was83.6 percent.

Calc.: C, 56.84; H, 3,18; N, 14.73;

Found: C, 56.81; H, 2.93; N, 14.50.

EXAMPLE 3 Preparation of 3-Bromo-6,7-methylenedioxycinnoline-4-ol About60 gm. (0.3 mole) of 6,7-methylenedioxycinnoline- 4-ol were added to asolution comprising 30 gm. (0.3 mole) of anhydrous potassium acetate in400 ml. of acetic acid. The resultant mixture was heated to reflux and asolution comprising 48 gm. (0.3 mole) of bromine in 100 ml. of aceticacid was added dropwise over a 2-hour period with stirring. After theaddition of the bromine was completed, the resultant reaction mixturewas stirred for an additional 30 minutes under reflux. The reactionmixture was allowed to cool to room temperature and then poured into 1liter of ice water. The resultant precipitate was recovered byfiltration and washed with 300 ml. of a S-percent sodium bicarbonatesolution. Recrystallization from an 80:20 mixture of dimethyl sulfoxideand water afforded 76 gm. of tan crystals of3-bromo-6,7-methylenedioxycinnoline-4-ol melting at 330 C. withdecomposition. The yield was 94.3 percent.

Analysis: C H BrN O Mol. Wt.: 269; Calc.: C, 40.17; H,

1.87; N, 10.41; Br, 29.70; Found: C, 40.31; H, 1.85; N, 10.60; Br,29.90.

EXAMPLE 4 Preparation of 6,7-Methylenedioxycinnoline-4-ol-3-carbonitrile About gm. (0.37 mole) of3-bromo-6,7-methylenedioxy-cinnoline-4-ol were added to a slurrycomprising 63 gm. (0.70 mole) of cuprous cyanide in 1,100 ml. of drydimethylformamide. The resultant mixture was heated to reflux for 4-5hours with stirring. Upon heating, the solution became homogenous. Oncompletion of the reaction, a heavy green precipitate formed. Theresultant reaction mixture was cooled to room temperature and pouredinto a solution comprising 160 gm. of ferric chloride, ml. ofconcentrated hydrochloric acid, and 250 ml. of water. The resultantmixture was heated to 60 C. for 15-20 minutes, poured over 2 liters ofcracked ice, and filtered. The yellow product was recrystallized from an80:20 mixture of dimethyl sulfoxide and water yielding 75 gm. (93.8percent) of 6,7-methylenedioxycinnoline-4-ol-3-carbonitrile as tancrystals which melted at 330-340 C. with decomposition.

Analysis: C l-hN O Mol. Wt.: 215;

Calc.: C, 55.82; H, 2.34; N, 19.53;

Found: C, 55.74; H, 3.04; N, 18.63.

EXAMPLE 5 Preparation of l-Ethyl-6,7-Methylenedioxy-4(1H)-Oxocinnoline-3- Carbonitrile About 70 gm. (0.36 mole) of6,7-methylenedioxycinnoline- 4-ol-3-carbonitrile were suspended in 500ml. of dry dimethylformamide. About 18 gm. (0.36 mole) of dry sodiumhydride (prepared by washing 50 percent sodium hydride in mineral oilwith Skelly B solvent) were added to the above-mentioned slurry withstirring. The mixture became homogenous as hydrogen was evolved. Afterthe cessation of hydrogen evolution, about 63 gm. (0.40 mole) of ethyliodide were added dropwise at room temperature with stirring. Stirringwas continued for 1 hour after the addition of the ethyl iodide wascompleted. The resultant reaction mixture was then heated to 100 C. for2 hours, cooled to room temperature, and poured into 1 liter of icewater and made acidic with hydrochloric acid. The precipitate which wasformed was removed by filtration and recrystallized from an 80:20mixture of dimethyl sulfoxide and water. Fifty grams, a yield of 62.5percent of 1- ethyl-6,7-methylenedioxy-4( l H )-oxocinnoline-3-carbonitrile as light tan crystals which melted at 268-269 C. wererecovered.

Analysis: C H N O Mol. Wt.: 243.22;

Calc.: C, 59.26; H, 3.73; N, 17.28;

Found: C, 59.05; H, 3.74; N, 17.10.

By following the foregoing procedure and substituting for the ethyliodide employed therein, a methyl, n-propyl, isopropyl, n-butyl, orallyl bromide, chloride, iodide or sulfate, the following compounds areobtained:

l -methyl-6,7-methylenedioxy-4( hH )-oxocinnoline-3-carbonitrilel-n-propyl-6,7-methylenedioxy-4( l H )oxocinnoline-3-carbonitrile 1-isopropyl-6,7-methylenedioxy-4( l H )-oxocinnoline-3-carbonitrilel-n-butyl-6,7-methylenedioxy-4( lH)-oxocinnoline-3-carbonitrilel-allyl-6,7-methylenedioxy-4( l H )-oxocinnoline- 3-carbonitrile EXAMPLE6 Preparation of l-Ethyl-6,7-Methylenedioxy-4( lH)- Oxocinnoline-3-Carboxylic acid About 23 gm. (0.095 mole) of l-ethyl-6,7-methylenedioxy-4( lH)-oxocinnoline-3-carbonitrile were added to a mixture of 200 ml. ofconcentrated hydrochloric acid and 200 ml. of acetic acid. The resultantreaction mixture was heated under reflux for 18 hours. The excess acidswere removed under vacuum, and the residue was taken up in ml. of aS-percent sodium bicarbonate solution. The resultant solution wastreated with 5 gm. of charcoal and filtered. The filtrate was madeacidic by the addition of hydrochloric acid and the resultingprecipitate was removed by filtration. Twenty-three grams, representinga yield of 9L6 percent, of l-ethyl-6,7-

methylenedioxy-4(lH)-oxocinnoline-3-carboxylic acid as light tancrystals which melted at 26l262 C. with decomposition were recovered.

Analysis: C, H, N O Mol. Wt.: 262;

Calc.: C, 54.96; H, 3.84; N, 10.68;

Found: C, 54.77; H, 3.84; N, 10.61.

By employing the foregoing hydrolysis procedure with the appropriatel-substituted nitrile, the following compounds are obtained:l-Methyl-6,7-methylenedioxy-4( lH)-oxocinnoline-3-carboxylic acid, m.p.308 C. (d.)

Analysis: Calc. for C H N O C, 53.23; H, 3.29; N, 11.29;

Found: C, 53.01; H, 3.27; N, 11.59. l-n-Propyl-6,7-methylenedioxy-4( 1H)-oxocinnoline-3-carboxylic acid, m.p. 22930 C.

Analysis: Calc. for C H N O C, 56.52; H, 4.38; N, 10.14;

Found: C,56.3l; H, 4.45; N, 10.36. 1-lsopropyl-6,7-methylenedioxy-4(lH)-oxocinnoline-3-carboxylic acid, m.p. 3045 C. (d.)

Analysis: Calc. for C, H, N O C, 56.52; H, 4.38; H, 10.14;

Found: C, 56.31; H, 4.29; N, 10.24. l-Allyl-6,7-methylenedioxy-4( 1H)-oxocinnoline-3-carboxylic acid, m.p. 2 l45 C.

Analysis: Calc. for C H N- O C, 56.93; H, 3.68; N, 10.22;

Found: C, 56.68; H, 3.86; N, 10.25.

1 -n-Butyl-6,7 methylenedioxy-4( 1H )-oxocinnoline- 3-carboxylic acid,m.p. 2lO-l 1C.

Analysis: Calc. for C H N O C, 57.93; H, 4.86; N, 9.65;

Found: C, 57.71; H, 4.98; N, 9.71.

The novel l-lower alkyl or alkenyl substituted-6,7-methylene-dioxy-4(1H)-oxocinnoline-3-carboxylic acid compounds of thisinvention and their salts, possess outstanding antimicrobial activity.Included among the organisms, pathogenic to warm-blooded animals,against which substantial activity has been demonstrated in vitro areMycoplasma gallisepticum and the Gram-negative bacteria Escherichiacoli, Salmonella dub/in, and Vibrio coli. These novel compounds havealso been found to be active against the plant pathogens pound againstthe test organism. The results of this test are compiled in Table II.The sodium salt was also administered by the oral route by incorporating200 gmJton thereof in chick feed fed continuously from the time ofhatch. Four groups of nine chicks each were provided with the medicatedfeed. Four groups of nine chicks each were fed the same ration butwithout the active compound. The eight groups of nine chicks each werechallenged by providing 50 ml. of a broth culture of S. typhimurium toeach group of chicks instead of drinking water with consumption beingcomplete within 24 hours. The number of survivors was determined on thetenth day. The oral activity of the compound against the test organismwas indicated by the difference in the number of survivors between thetreated and untreated groups. Table lll shows the results of this test.

TABLE II Antimicrobial Activity of 1Ethyl-6,7-methylenedioxy-4( 1 Hoxocinnoline-3-carboxylic acid, Sodium Salt, lnjected AntimicrobialActivity of 1-Ethyl-6,7-methylenedioxy-4( 1H)- oxocinnoline-3-carboxylicacid, Sodium Salt, incorporated in the Feed of Newly Hatched ChicksErwinia amylovora, the causative agent of fire blight of appliessurvivors( 10 Days)/ and pears, Xanthomonns phaseoli, the causativeagent of com- Replcme Treatmem Tesed mon bacterial blight of beans, andAster yellows, a M ycoplasma disease. 1 1. 7

Representative novel compounds of this invention (as their hy di yisodium salts) have the following minimum inhibitory concentrations(expressed micrograms per milliliter) against the sodium 200 foregomgorganisms In the conventional tube dilution test. gJmn TABLE I.IN VITROANTIMICROBIAL ACTIVITY Minimum inhibitory concentration, mcg. ml. whentested with M. aalli Compound E. coli S. dublin V. coli septicuml-ethyl-G, 7methylenedioxy-i(1H)-oxocinnolinc-3carboxylic acid, sodiumsalt 12. 5 6. 25 25. 0 3.12 l-isopropyl-G,7-niethylenedioxy-4(1H)-oxocinnoline-3-carboxyl1c acid, sodium salt"25.0 12. 5 50.0 25. 0 l-allyl-G, T-methyleneOXY-KIH)-OXOClI1Il0llH9-3-C81'DOXY]l0 acid,

sodium salt 50.0 12. 5 50. 0 50.0

Also, the l-lower alkyl or alkenyl substituted-6,7- g g gmethylenedioxy-4( lH)-oxocinnoline-3-carboxylic acids, and 4 1 theirsalts, are effective therapeutic agents for microbial infecm 19/36 tionsin warm-blooded animals. Moreover, they are effective by eitherparenteral or oral administration, as demonstrated 1 I f d controls 0/9by tests with young chicks. For example, the sodium salt of 1- 2a 0/9ethyl-6,7-methylenedioxy-4( 1H )-oxocinnoline-3-carboxylic u 3/ 9 40 U9acid was in ected into the neck of day-old chicks at a level of Totals4/36 approximately 50 mg. per chick. Two groups of five chicks each wereso treated. Two groups of five chicks each were included as untreatedcontrols. The four groups of five chicks each were immediatelychallenged with 0.1 ml. of a Salmonel- Ia typhimurium culture givenintramuscularly. On the sixth day the number of survivors was determinedfor each group. The number of survivors indicated the activity of thecom- Furthermore, the injection of 1-ethyl-6,7-methylenedioxy-41H)-oxocinnoline-3-carboxylic acid, sodium salt, into the neck of6-week-old broiler chickens, weighing about 600 gm., protected thetreated chickens from challenge doses of E. coli. Ten groups of 10chickens each were used in the test. Six of the 10 groups were injectedsubcutaneously with the active compound. The other four groups served asinfected controls. Dosage levels of 50, 100, and 200 mg./kg. wereemployed. The air sac of each chicken was injected immediately aftertreatment with 0.5 ml. of a 10 dilution of a 24-hour tryptose brothculture of an E. coli strain, one-half of the groups getting the Grossstrain, the other half the Glantz strain. Seven days after treatment,the surviving chickens were sacrificed and autopsied. Each chicken wasexamined for air sac lesions, pericarditis, and perihepatitus. The meanlesion score for each group was determined by assigning the arbitraryscale to each chicken: 4 death before autopsy; 3 air sac lesion pluspericarditis plus perihepatitis; 2 air sac lesion plus eitherpericarditis or perihepatitis; l air sac lesion; 0 no lesion. The totalfor the group was summed and divided by the number of chickens in thegroup. A mean lesion score of 4.0 signifies all chickens died before theend of the test, while a score of 0.0 means there were no deaths and nolesions were observed at necropsy. The difference in the magnitude ofthe mean lesion scores between the treated and untreated chickensindicates the activity of the compound against the test organism. TableIV lists the results of this test.

TABLE IV Antiinfective Activity of 1-Ethyl-6,7-Methylenedioxy-4( 1H)-oxocinnoline-3-carboxylic acid, Sodium Salt, injected subcutaneouslyinto Six-Week-Old Chickens The sodium salt ofl-ethyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carboxylic acid wasdissolved in water is a concentration of 1,000 p.p.m. and 0.0175percent, weight/volume, of Toximul R and 0.0175 percent, weight/volume,of Toximul S (proprietary blends of non-ionic and anionic wetting agentssupplied by Stepan Chemical Co., Northfield, 111.) were added to thesolution. The resulting solution was sprayed onto orchard-grown,6-year-old Bartlett pear trees artificially inoculated with Erwiniaamylovora (fire blight) until the leaves, branches, and trunk werewetted to the point of run-off. Expression of the symptoms of fireblight disease was arrested.

A stable liquid concentrate of l-ethyl-6,7-methylenedioxy-4ll-l)-oxocinnoline-3-carboxylic acid, sodium salt can be prepared bydissolving from about 10 to about 50 percent weight/volume, of theactive agent in deionized water and adding thereto from about 1 to about10 percent, weight/volume, of a polyoxyethylene (10 mol.) alkyl phenolwetting agent. From about 1 to about 10 percent, weight/volume, of asalt of a lignin sulfonate is generally included in the formulation toserve as a chelating agent for heavy metal ions found in the wateravailable as a diluent in many localities. A representative formula iscomprised of:

l-ethyl-6,7-methylenedioxy- 4( lH)-oxocinnoline-3- carboxylic acid,sodium salt 480 grams Triton X- l 00 [polyoxyethylene 10 mol.)octylphenol, wetting agent] grams Polyfon H (sodium lignin sulfonate)100 grams Deionized Water Q.S. 1000 ml.

The above-described formulation contains 4 lbs. of the active agent pergallon of concentrate; a popular concentration employed in agriculturalchemicals formulations. This liquid concentrate is diluted withavailable water to a concentration of active ingredient appropriate forthe intended use. For example, to make a spray solution containing aboutl,000 p.p.m. of the active agent one quart of the above-describedconcentrate is added to about gallons of water. The resulting solutionis applied with a pressure sprayer at from about 20 to about 100 psi. tothe plants to be treated until all of the foliage and stems are wettedto the point of run-off.

The l-lower alkyl or alpenylsubstituted-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carboxylic acids,and their salts, have a low level of mammalian toxicity. Thus, forexample, no deaths are encountered when the sodium salt of l-ethyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carboxylic acid is administered atlevels as high as 200 mg./kg. intraperitoneally or 500 mg./kg. orally tomice.

The novel compounds of this invention and their salts are effective incombating microbial infections in warm-blooded animals when administeredin total daily amounts of from about 25 mg. to about 500 mgJkg. of bodyweight.

Because of their greater water solubility, chemical stability,substantial eflicacy, ease of preparation, and amenability toincorporation in various pharmaceutical dosage forms and medicatedfeeds, the use of the alkali metal and ammonium salts of the l-loweralkyl or alpenyl substituted-6,7- methylenedioxy-4(1I-1)-oxocinnolinecarboxylic acids are preferred in the practice of this invention.

In a preferred embodiment of the present invention, 1-ethyl-6,7-methylenedioxy-4( 1H )-oxocinnoline-3-carboxylic acid as asalt is administered orally in a suitable feed in which the compound ispresent in an amount of from about 25 to about 2,500 grams per ton oftotal feed, the exact concentration depending on the amount required toprovide for the ingestion of an efiective dosage of the active agentwhen normal amounts of feed are consumed. The addition of the compoundsof this invention to feed is preferably accomplished by preparing anappropriate feed premix and incorporating the premix into the completeration. Alternatively, an intermediate concentrate or feed supplementcontaining the compound can be blended into the feed.

The preparation of a suitable feed premix can be effected by grindingthe compounds of this invention to a powder and admixing with a suitablecarrier such as alfalfa grits, solvent-extracted soybean feed, cornmeal, exfoliated hydrobiotite, and like carriers. The premix so preparedis then admixed with whatever feed ration is being fed to the animal atthe time of administering the compounds of this invention. The feedpremix can first be diluted with a feed supplement or feed concentrateto a desired concentration of the active compound, and the medicatedsupplement or concentrate can either be fed concurrently with theremainder of the ration or can be mixed into the final feed.

An alternative procedure for preparing the premix comprises dissolvingthe active compound in a suitable solvent such as water, or preparing aslurry of the compound in an appropriate vehicle such as an ediblevegetable oil or an edible glycol, and adding such solution or slurry tothe premix carrier by spraying onto the carrier with suitable mixing.

The l-lower alkyl or alpenyl substituted-carboxylic acids and theirsalts can be administered as a single dose or in divided doses either inparenteral or oral dosage form. When a parenteral dosage form isindicated, the active agent can be dissolved in an aqueous vehicle in aconcentration of from (1H)-oxocinnoline-3-carboxylic acid can beprocessed into tablets suitable for oral administration by the followingprocedure: 5.0 gm. of 1-ethyl-6,7-methylenedioxy-4(lH)-oxocinnoline-S-carboxylic acid are mixed with 22.0 gm. of lactose,2.9 gm. of starch, and 0.1 gm. of magnesium stearate, and the mixture ispressed into tablets of such size that each tablet contains 50 mg. ofthe active agent.

What is claimed is:

l. A compound of the class consisting of compounds of the followingformula:

C-OH W Wherein R is a member of the group consisting of methyl, ethyl,n-propyl, isopropyl, n-butyl, vinyl and allyl, and the cationic saltsthereof formed with an alkali metal, or alkaline earth metal, ammonia,triethylamine, or pyrrolidine.

2. A compound according to claim 1 wherein the compound isl-ethyl-6,7-methylenedioxy-4( lH)-oxocinnoline-3-carboxylic acid.

3. A compound according to claim 1 wherein the compound isl-methyl-6,7-methylenedioxy-4( 1H )-oxocinnoline-3-carboxylic acid.

4. A compound according to claim 1 wherein the compound isl-vinyl-6,7-methylenedioxy-4( 1H )-oxocinnoline-3-carboxylic acid.

5. A compound according to claim 1 wherein the compound isl-(n-propyl)-6,7-methylenedioxy-4( 1H)-oxocinnoline-3- carboxylic acid.

6. A compound according to claim 1 wherein the compound isl-allyl-6,7-methylenedioxy-4( lH)-oxocinnoline-3-carboxylic acid.

7. A compound according to claim 1 wherein the compound isl-isopropyl-6,7-methylenedioxy-4( lH)-oxocinnoline-3-carboxylic acid.

8. A compound according to claim 1 wherein the compound isl-(n-butyl)-6,7-methylenedioxy-4( lH)-oxocinnoline-3-carboxylic acid.

9. A method for the preparation of a compound of the class of compoundsof the following formula:

II 0 O OH 2 w N 0 III Wherein R is a member of the group consisting ofmethyl, ethyl, n-propyl, isopropyl, n-butyl and allyl which comprises:

a. catalytically reducing 2-nitro-4,5-mcthylenedioxyacetophenone to2-amino-4',5-methylenedioxyacetone.

b. diazotizing 2-amino-4',5'-methylenedioxyacetophenone with nitrousacid and cyclizing to yield 6,7-

methylenedioxycinnoline-4-ol c. treating6,7-methylenedioxycinnoline-4-ol with bromine to produce3-bromo-6,7-methylene-dioxycinnoline-4-ol d. treating3-bromo-6,7-methylenedioxycinnoline-4-ol with cuprous cyanide to form6,7-methylenedioxy-cinnoline- 4-ol-3-carbonitrile e. alkylating6,7-methylenedioxycinnoline-4-ol-3-carbonitrile with an alkyl oralkylene bromide, chloride, iodide or sulfate to form a l-lower alkyl oralkylene substituted-o,7-methylenedioxy-4(lH)-oxocinnoline-3-carbonitrile of the following formula:

m n E Wherein R is a member of the group consisting of methyl, ethyl,n-propyl, isopropyl, n-butyl and ally] and:

f. hydrolyzing the l-lower alkyl or alkylene substituted-6,7-

methylenedioxy-4( 1H)-oxocinnoline-3-carbonitrile l0.3-Bromo-6,7-methylenedioxycinnoline-4-ol l 1.6,7-methylenedioxycinnoline-4-ol-3-carbonitrile 12. A compound of theclass consisting of compounds of the following formula:

Wherein R is a member of the group consisting of methyl,

ethyl, n-propyl, isoproply, n-butyl, B-hydroxyethyl and allyl.

13. A compound according to claim 12 wherein the compound isl-methyl-6,7-methylenedioxy-4(1H)-oxocinnoline- 3-carbonitrile.

14. A compound according to claim 12 wherein the compound islet.hyl-6,7-methylenedioxy-4( lH)-oxocinnoline-3- carbonitrile.

15. A compound according to claim 12 wherein the compound isl-(n-propyl)-6,7-methylenedioxy-4(lH)-oxocinnoline-3-carbonitrile.

16. A compound according to claim 12 wherein the compound isl-isopropyl-6,7-methylenedioxy-4( lH)-oxocinnoline-3-carbonitrile.

17. A compound according to claim 12 wherein the compound isl-isopropyl-6,7-methylenedioxy-4(lH)-ox0cinnoline-3-carbonitrile.

18. A compound according to claim 12 wherein the compound isl-B-hydroxylethyl-6,7-methylenedioxy-4(lH)-oxocinnoline-3-carbonitrile.

19. A compound according to claim 12 wherein the compound isl-allyl-6,7-methylenedioxy-4(ll-l)-oxocinnoline-3- carbonitrile.

t l =t

2. A compound according to claim 1 wherein the compound is1-ethyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carboxylic acid.
 3. Acompound according to claim 1 wherein the compound is1-methyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carboxylic acid.
 4. Acompound according to claim 1 wherein the compound is1-vinyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carboxylic acid.
 5. Acompound according to claim 1 wherein the compound is1-(n-propyl)-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carboxylic acid. 6.A compound according to claim 1 wherein the compound is1-allyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carboxylic acid.
 7. Acompound according to claim 1 wherein the compound is1-isopropyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carboxylic acid. 8.A compound according to claim 1 wherein the compound is1-(n-butyl)-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carboxylic acid. 9.A method for the preparation of a compound of the class of compounds ofthe following formula:
 10. 3-Bromo-6,7-methylenedioxycinnoline-4-ol 11.6,7-methylenedioxycinnoline-4-ol-3-carbonitrile
 12. A compound of theclass consisting of compounds of the following formula:
 13. A compoundaccording to claim 12 wherein the compound is1-methyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carbonitrile.
 14. Acompound according to claim 12 wherein the compound is1-ethyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carbonitrile.
 15. Acompound according to claim 12 wherein the compound is1-(n-propyl)-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carbonitrile.
 16. Acompound according to claim 12 wherein the compound is1-isopropyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carbonitrile.
 17. Acompound according to claim 12 wherein the compound is1-isopropyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carbonitrile.
 18. Acompound according to claim 12 wherein the compound is 1-Beta-hydroxylethyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carbonitrile. 19.A compound according to claim 12 wherein the compound is1-allyl-6,7-methylenedioxy-4(1H)-oxocinnoline-3-carbonitrile.